Pyruvoyl-dependent histidine decarboxylases. Mechanism of cleavage of the proenzyme from Lactobacillus buchneri

When Lactobacillus buchneri was grown in the presence of [hydroxyl-18O]serine and pyridoxamine, no 18O was found in its histidine decarboxylase (HisDCase). However, when pyridoxamine was omitted from the growth medium, the labeled serine was incorporated into the HisDCase without dilution. Internal serine residues of the enzyme contained 18O only in their hydroxyl group, while the COOH-terminal serine of the beta chain of HisDCase contained equal amounts of 18O in both its hydroxyl and carboxyl group. This enzyme, like the HisDCase from Lactobacillus 30a (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977), therefore, arises by nonhydrolytic serinolysis of its proenzyme. This result, together with comparative sequence data (Huynh, Q. K., and Snell, E. E. (1985) J. Biol. Chem. 260, 2798-2803), makes it highly probable that all of the pyruvoyl-dependent HisDCases arise by a similar mechanism from inactive proenzymes.     

Pyruvoyl-dependent histidine decarboxylases. Comparative sequences of cysteinyl peptides of the enzymes from Lactobacillus 30a, Lactobacillus buchneri, and Clostridium perfringens

The two cysteinyl residues present in histidine decarboxylase from Lactobacillus 30a differ greatly in reactivity. One (class 1) reacts readily in the native state with dithiobis-(2-nitrobenzoate) with complete loss of enzyme activity; the other (class 2) reacts only after denaturation of the enzyme (Lane, R. S., and Snell, E. E. (1976) Biochemistry 15, 4175-4179). These differences in reactivity permitted use of covalent (disulfide) chromatography to isolate separate peptides that contain these two residues. Sequence analysis showed that the class 1 cysteinyl residue is at position 147 in a hydrophilic portion of the alpha chain (Huynh, Q. K., Recsei, P. A., Vaaler, G. L., and Snell, E. E. (1984) J. Biol. Chem. 259, 2833-2839), while the class 2 cysteinyl residue is present at position 71, adjacent to a hydrophobic portion of the same chain. Cysteinyl peptides identical with or homologous to the class 2 cysteinyl peptide of the Lactobacillus 30a enzyme were isolated from the alpha subunits of histidine decarboxylases from Lactobacillus buchneri and Clostridium perfringens, respectively. The L. buchneri enzyme also contained a peptide homologous to the class 1 cysteinyl peptide from Lactobacillus 30a. However, no corresponding peptide was present in the enzyme from C. perfringens, in which the second cysteinyl residue of the alpha chain occupies position 3, very near the essential pyruvoyl residue. This enzyme, unlike those from Lactobacillus 30a or L. buchneri, also contains one cysteinyl residue in its beta chain. Although Cys 147 is an active site residue in histidine decarboxylase from Lactobacillus 30a, the absence of a corresponding residue in the C. perfringens enzyme confirms previous indications (Recsei, P. A., and Snell, E. E. (1982) J. Biol. Chem. 257, 7196-7202) that this SH group is not essential for decarboxylase action.     

Site-directed alteration of the active-site residues of histidine decarboxylase from Clostridium perfringens

To clarify the mechanism of biogenesis and catalysis by the pyruvoyl-dependent histidine decarboxylase (HisDCase) from Clostridium perfringens, 12 mutant genes encoding amino acid substitutions at the active site of this enzyme were constructed and expressed in Escherichia coli. The resulting mutant proteins were purified to homogeneity, characterized, and subjected to kinetic analysis. The results (a) exclude all polar amino acid residues in the active site except Glu-214 as donor of the proton that replaces the carboxyl group of histidine during decarboxylation and, since E214I and E214H are nearly inactive, indicate that Glu-214 is the essential proton donor; (b) demonstrate the importance to substrate binding of hydrophobic interactions between Phe-98, Ile-74, and the imidazole ring of histidine, and of hydrogen bonding between Asp-78 and N2 of the substrate; and (c) demonstrate a significant unidentified role for Glu-81 in the maintenance of the active-site structure. The proposed roles of these amino acid residues are consistent with those assigned on the basis of crystallographic evidence to the corresponding residues at the active site of the related HisDCase from Lactobacillus 30a [Gallagher, T., Snell, E. E., & Hackert, M. L. (1989) J. Biol. Chem. 264, 12737-12743]. Of the residues altered, only Ser-97 was essential for the autocatalytic serinolysis reaction by which this HisDCase, (alpha beta)6, is derived from its inactive, pyruvate-free precursor, proHisDCase, pi 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histidine decarboxylase of Lactobacillus 30a. Hydroxylamine cleavage of the -seryl-seryl- bond at the activation site of prohistidine decarboxylase

Conversion of the pi subunit of prohistidine decarboxylase to the alpha beta subunits of the active enzyme proceeds by a nonhydrolytic, monovalent cation-dependent, serinolysis reaction in which the hydroxyl oxygen of serine 82 of the pi chain is incorporated into the carboxyl group at the COOH terminus (serine 81) of the beta chain. Serine-82 becomes the pyruvate residue at the NH2 terminus of the alpha chain (Recsei, P.A., Huynh, Q. K., and Snell, E.E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977). The unusual reactivity of this particular -Ser-Ser- bond is demonstrated by its sensitivity to 1 M hydroxylamine, which cleaves the native proenzyme under mild conditions (pH 8.0, 37 degrees C) to yield a modified beta chain with serine hydroxamate at the COOH terminus (Ser-81) and a modified alpha chain containing serine (Ser-82 of the proenzyme) rather than pyruvate at the NH2 terminus. Neither an -Asn-Gly- bond nor other -Ser-Ser- bonds in the proenzyme were cleaved under these conditions. The reaction also did not occur with the denatured enzyme or with model peptides, indicating that the enhanced reactivity is a result of the particular conformation at this position in the native protein. The reaction with the native proenzyme proceeded optimally at pH 7.5-8.0 with a half-time (30 min) substantially less than that (3.5-4.5 h) required for the activation reaction and was not increased in rate by addition of K+. Correspondingly, preincubation of the proenzyme at pH 8.0 in the absence of both hydroxylamine and K+ modestly increased the rate of activation when K+ was subsequently added. Although these findings do not exclude other mechanisms, they are all consistent with and most easily explained by rearrangement of the pi chain to form an internal ester intermediate prior to the beta-elimination that occurs during activation to yield the alpha and beta chains of the mature enzyme.     

Escherichia coli S-adenosylmethionine decarboxylase. Subunit structure, reductive amination, and NH2-terminal sequences

S-Adenosylmethionine decarboxylase is one of a small group of enzymes that use a pyruvoyl residue as a cofactor. Histidine decarboxylase from Lactobacillus 30a, the best studied pyruvoyl-containing enzyme, has an (alpha beta)6 subunit structure with the pyruvoyl moiety linked through an amide bond to the NH2-terminal of the larger alpha subunit (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977). To examine potential structural analogies between the two enzymes, we have isolated and partially characterized S-adenosylmethionine decarboxylase. The purified enzyme comprises equimolar amounts of two subunits of Mr = 14,000 and 19,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and has a native molecular weight of 136,000 (by gel filtration). Approximately 4 mol of [methyl-3H] adenosylmethionine are incorporated per mol of enzyme (Mr = 136,000) when the enzyme is inactivated with this substrate and NaCNBH3. These data suggest an (alpha beta)4 structure with 1 pyruvoyl residue for each alpha beta pair. The two subunits have been separated by reversed-phase high performance liquid chromatography after reduction and carboxymethylation. The smaller subunit (beta) has a free amino terminus. The amino terminus of the larger subunit (alpha) appears to be blocked by a pyruvoyl group; this subunit can be sequenced only after this group is converted to an alanyl residue by reduction with sodium cyanoborohydride in the presence of ammonium acetate. This work suggests that S-adenosylmethionine decarboxylase is structurally much more similar to histidine decarboxylase than previously thought.     

Histidine decarboxylase of Lactobacillus 30a. Sequences of the overlapping peptides, the complete alpha chain, and prohistidine decarboxylase

The complete amino acid sequence of the alpha chain of histidine decarboxylase of Lactobacillus 30a has been established by isolation and analysis of the eight methionine-containing tryptic peptides of this chain. These peptides provide the overlaps required to order all nine peptides derived by complete cyanogen bromide cleavage of the alpha chain (Huynh, Q.K., Vaaler, G.L., Recsei, P.A., and Snell, E.E. (1984) J. Biol. Chem. 259, 2826-2832). Ordering of six of the latter peptides was confirmed by isolation and analysis of four peptides derived by incomplete cyanogen bromide cleavage. The alpha chain is composed of 226 residues and has a molecular weight of 24,892 calculated from the sequence. These results and the previously determined sequence of the beta chain (Vaaler, G.L., Recsei, P.A., Fox, J.L., and Snell, E.E. (1982) J. Biol. Chem. 257, 12770-12774) establish the complete amino acid sequence of the enzyme and of the pi chain of prohistidine decarboxylase. The latter is composed of 307 amino acids and has a calculated molecular weight of 33,731. Four segments of the pi chain sequence are repeated. The bond between Ser-81 and Ser-82 that is cleaved during proenzyme activation is in an uncharged portion of the sequence that is rich in serine and threonine residues and is predicted to be part of a beta sheet structure.     

Pyridoxal phosphate-dependent histidine decarboxylases. Cloning, sequencing, and expression of genes from Klebsiella planticola and Enterobacter aerogenes and properties of the overexpressed enzymes

The hdc genes encoding the inducible pyridoxal-P-dependent histidine decarboxylase (HisDCase) of Klebsiella planticola and Enterobacter aerogenes were isolated, sequenced, and expressed in Escherichia coli under control of the lac promoter, and the overproduced enzymes were purified to homogeneity from the recombinant host. Formation of inclusion bodies during synthesis of the E. aerogenes enzyme was avoided by cooling the culture and inducing at 25 degrees C. The cloned enzymes were produced in amounts three to four times those present in the fully induced native hosts and were identical in properties to those isolated earlier (Guirard, B. M., and Snell, E. E. (1987) J. Bacteriol. 169, 3963-3968). The two enzymes showed 85% sequence identity and also showed 80% sequence identity with the previously sequenced (Vaaler, G. L., Brasch, M. A., and Snell, E. E. (1986) J. Biol. Chem. 261, 11010-11014) HisDCase of Morganella morganii. Nevertheless, antibodies to the M. morganii HisDCase do not cross-react with these enzymes suggesting that the regions of amino acid variations are located on the outer surface of the proteins. All three HisDCases are the same length (377 amino acid residues); encoded N-terminal methionine was completely removed in each case. These closely related pyridoxal-P enzymes show no sequence homology with the pyruvoyl-dependent HisDCases of Gram-positive bacteria.     

Three-dimensional structure of a ubiquitin-conjugating enzyme (E2).

The x-ray crystal structure of a recombinant ubiquitin-conjugating enzyme (E2) encoded by the UBC1 gene of the plant Arabidopsis thaliana has been determined with the use of multiple isomorphous replacement techniques and refined at 2.4-A resolution by simulated annealing and restrained least-squares. This E2 is an alpha/beta protein, with four alpha-helices and a four-stranded antiparallel beta-sheet. The NH2 and COOH termini, which may be important for interaction with other enzymes and substrates in the ubiquitin-conjugation pathway, are on the opposite side of the molecule from the cysteine residue that binds to the COOH terminus of ubiquitin. This structure should now allow for the rational analysis of E2 function by in vitro mutagenesis and facilitate the effective design of E2s with unique specificities or catalytic functions.     

Histidine decarboxylase of Lactobacillus 30a. Sequences of the cyanogen bromide peptides from the alpha chain

The alpha chain of histidine decarboxylase contains eight internal methionine residues. After reductive amination to convert the NH2-terminal pyruvoyl residue to an alanyl residue and cyanogen bromide treatment, 13 pure peptides were isolated. Four of these are incomplete cleavage products. The sequence of each of the remaining nine peptides was established by automated and manual degradation of the intact peptides and of smaller peptides obtained from tryptic, chymotryptic, and staphylococcal protease digests of the cyanogen bromide peptides. These results, together with the data on overlapping peptides reported in the accompanying paper (Huynh, Q. K., Recsei, P. A., Vaaler, G. L., and Snell, E. E. (1984) J. Biol. Chem. 259, 2833-2839), establish the complete amino acid sequence of the alpha chain.     

Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains

The enzymatic domains of the avian retrovirus polymerase (pol) gene have been mapped by the use of peptide antibodies and COOH-terminal amino acid analysis. The processed pol beta polypeptide is cleaved in vivo to yield alpha and pp32. Rabbit antibodies were directed against synthetic peptides whose sequence was deduced from the known pol sequence of Rous sarcoma virus, Prague C (Schwartz, D.E., Tizard, R., and Gilbert, W. (1983) Cell 32, 853-869). The RNase H active site of pol was located in the NH2-terminal region of the alpha DNA polymerase subunit. The COOH terminus of the alpha subunit was found to be immediately adjacent to the NH2 terminus of the pp32 pol protein. COOH-terminal amino acid analysis of pp32 revealed that this protein is also processed. From the deduced amino acid sequence of pol, it appears likely that pol encodes an additional 4100-dalton polypeptide located at its extreme COOH terminus. The enzymatic domains on beta appear to map in the following order: RNase H-DNA polymerase-DNA endonuclease. Hydrophilicity analysis and secondary structure predictions of wild type Rous sarcoma virus pol products and mutated pp32 possessing single amino acid changes permit further structural evaluation of the multifunctional pol protein.     

The alpha subunit of meprin A. Molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the alpha and beta subunits.

Meprin A, a membrane-bound oligomeric metalloendopeptidase, contains two different subunits, alpha and beta. We report here the cloning and sequencing of the alpha subunit cDNA. The translated polypeptide consists of 760 amino acids, including a preprosequence (77 amino acids) that precedes the NH2 terminus of the purified enzyme. The next 198 amino acids constitute the "astacin family" protease domain, which includes the astacin family signature sequence, HE(L,I)XHXXGFXHE(Q,H)XRXDRDX(Y,H)(V,I)X(I,V). An immunoglobulin/major histocompatibility complex protein signature was found at the end of the protease domain. At the COOH terminus of the alpha subunit, there is an epidermal growth factor-like domain, followed by a transmembrane domain, and six additional amino acids. Ten potential glycosylation sites have been identified, and at least three of those sites are glycosylated. Northern blot analyses of kidney tissue from C57BL/6 and C3H/He mice indicate that variations in meprin A activity in these strains reflect differences in the levels of the alpha subunit mRNA. Several internal peptide sequences obtained from the beta subunit indicate that it is approximately 50% identical to the alpha subunit. Furthermore, NH2-terminal sequence analyses (39 residues) indicate that rat and mouse alpha are 79% identical, rat and mouse beta are 74% identical, and that alpha and beta subunits for both species are 47% identical. These data indicate that alpha and beta are closely related products of divergent evolution.     

Heavy riboflavin synthase of Bacillus subtilis. Primary structure of the beta subunit

Heavy riboflavin synthase is a 1,000,000-Da protein catalyzing the last two reactions of riboflavin biosynthesis. The enzyme complex consists of 60 beta subunits (Mr = 16,200) and approximately three alpha subunits (Mr = 23,000). beta subunits were isolated and cleaved with cyanogen bromide. Fragments were isolated and further digested with trypsin and staphylococcal protease. Peptides were isolated by high performance liquid chromatography. Sequences were determined by automated liquid-phase Edman degradation. The complete sequence of the beta subunit (154 amino acids) was established by direct sequencing of the NH2 terminus, sequencing of overlapping peptides, and carboxypeptidase degradation of the COOH terminus. The sequence shows no detectable homologies to other proteins. A computer prediction of secondary structure elements indicates 34% alpha helix and 30% beta sheet.     

Conservation of human fibrinogen conformation after cleavage of the B beta chain NH2 terminus

Human fibrinogen exposed to protease III from Crotalus atrox venom is cleaved near the NH2 terminus of the B beta chain yielding a species of Mr 325,000 (Fg325) with impaired thrombin clottability. The derivative was compared with intact fibrinogen in a number of ways to determine whether the functional defect resulted from a conformational change or from the loss of a polymerization site. NH2-terminal amino acid sequencing of isolated A alpha, B beta, and gamma chains showed that Fg325 contained intact A alpha and gamma chains, but differed from fibrinogen by the absence of the first 42 residues of the B beta chain. Fibrinopeptide A was present and was cleaved at the same rate in both fibrinogen and Fg325. The rate and extent of A alpha and gamma cross-linking by factor XIIIa was also indistinguishable. In contrast, the thrombin-catalyzed coagulation of Fg325 was 46% less in extent and 180-fold slower than observed for intact fibrinogen. A conformational comparison of Fg325 and fibrinogen was made using immunochemical and spectroscopic approaches. Antisera specific for different regions of the fibrinogen molecule were used to characterize the epitopes in Fg325. The only significant differences were found in the NH2-terminal region of the B beta chain, probed with antiserum to B beta 1-118. The conformational similarity of Fg325 and fibrinogen was confirmed by the identity of both near and far UV CD spectra of the two proteins. Structural, functional, and immunochemical results imply that cleavage of 42 NH2-terminal residues from the B beta chain is not accompanied by a measurable conformational change. The residues of this B beta chain segment, which are evidently located on the surface of the molecule, in conjunction with the NH2-terminal part of the A alpha chain appear to play an important role in the expression of a fibrin polymerization site.     

Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Nucleotide sequence of the hdc gene and the corresponding amino acid sequence

The nucleotide sequence of a 1.3-kilobase NaeI fragment from Morganella morganii AM-15 that contains the gene for histidine decarboxylase has been determined. The gene was initially identified among total chromosomal digests using a mixed sequence oligonucleotide probe corresponding to amino acids 11-16 of histidine decarboxylase and then cloned on a 5.5-kilobase PstI fragment. The structural gene contains 1131 nucleotides and encodes 377 amino acids with the sequence: (sequence: in text). The independently determined NH2-terminal sequence of this enzyme (Tanase, S., Guirard, B. M., and Snell, E. E. (1985) J. Biol. Chem. 260, 6738-6746) and the amino acid sequences of two tryptic peptides reported in the accompanying paper (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009) are localized in the sequence presented here; the lysine that binds pyridoxal phosphate is situated at residue 232, whereas the serine that binds the adduct formed between pyridoxal phosphate and the inhibitor alpha-fluoromethylhistidine is positioned at residue 322.     

Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins

The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.     

Reaction of Lactobacillus histidine decarboxylase with L-histidine methyl ester

L-Histidine methyl ester inactivates histidine decarboxylase in a time-dependent manner. The possibility was considered that an irreversible reaction between enzyme and inhibitor occurs [Recsei, P. A., & Snell, E. E. (1970) Biochemistry 9, 1492-1497]. We have confirmed time-dependent inactivation by histidine methyl ester and have investigated the structure of the enzyme-inhibitor complex. Upon exposure to either 8 M guanidinium chloride or 6% trichloroacetic acid, unchanged histidine methyl ester is recovered. Formation of the complex involves Schiff base formation, most likely with the active site pyruvyl residue [Huynh, Q. K., & Snell, E. E. (1986) J. Biol. Chem. 261, 4389-4394], but does not involve additional irreversible covalent interaction between inhibitor and enzyme. Complex formation is a two-step process involving rapidly reversible formation of a loose complex and essentially irreversible formation of a tight complex. For the formation of the tight complex, Ki = 80 nM and koff = 2.5 X 10(-4) min-1. Time-dependent inhibition was also observed with L-histidine ethyl ester, L-histidinamide, and DL-3-amino-4-(4-imidazolyl)-2-butanone. No inactivation was observed with glycine methyl ester or histamine. We propose that in the catalytic reaction the carboxyl group of the substrate is in a hydrophobic region. The unfavorable interaction between the carboxylate group and the hydrophobic region facilitates decarboxylation [Crosby, J., Stone, R., & Liehard, G. E. (1970) J. Am. Chem. Soc. 92, 2891-2900]. With histidine methyl ester this unfavorable interaction is no longer present; hence, there is tight binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of expression mutants and monoclonal antibodies to map the erythrocyte Ca2+ pump.

Deletion and truncation mutants of the human erythrocyte Ca2+ pump (hPMCA4b) were expressed in COS-1 cells. The reactivity patterns of these mutants with seven monoclonal antibodies were examined. Of the seven, six (JA9, JA3, 1G4, 4A4, 3E10 and 5F10) react from the cytoplasmic side. JA9 and JA3 reacted near the NH2 terminus and the COOH terminus of the molecule, respectively. 5F10 and 3E10 recognized portions of the large hydrophilic region in the middle of the protein. The epitopes of 1G4 and 4A4 were discontinuous and included residues from the long hydrophilic domain and residues between the proposed transmembrane domains M2 and M3. Antibody 1B10, which reacts from the extracellular side, recognized the COOH-terminal half of the molecule. These results show that the NH2 terminus, the COOH terminus, the region between M2 and M3, and the large hydrophilic region are all on the cytoplasmic side. This means that there are an even number of membrane crossings in both the NH2-terminal and the COOH-terminal halves. Between residues 75 and 300 there must be at least two membrane crossings, and there are at least two membrane crossings in the COOH-terminal half of the molecule.     

Localization of the exposed N-terminal region of the B800-850 alpha and beta light-harvesting polypeptides on the cytoplasmic surface of Rhodopseudomonas capsulata chromatophores.

Proteinase K and trypsin were used to determine the orientation of the light-harvesting B800-850 alpha and beta polypeptides within the chromatophores (inside-out membrane vesicles) of the mutant strain Y5 of Rhodopseudomonas capsulata. With proteinase K 7 amino acid residues of the B800-850 alpha polypeptide were cleaved off up to position Trp-7--Thr-8 of the N terminus, and 11 residues were cleaved off up to position Leu-11-Ser-12 of the beta chain N terminus. The C termini of the B800-850 alpha and beta polypeptides, including the hydrophobic transmembrane portions, remained intact. It is proposed that the N termini of the alpha and beta subunits, each containing one transmembrane alpha-helical span, are exposed on the cytoplasmic membrane surface and the C termini are exposed to or directed toward the periplasm.     

The sequence of a cloned cDNA for the beta subunit of bovine thyrotropin predicts a protein containing both NH2-and COOH-terminal extensions

Cloned cDNAs containing sequences coding for the beta subunit of bovine thyrotropin have been identified. The complete nucleotide sequence of the largest of the beta subunit cDNA inserts has been determined. This cDNA contains 35 nucleotides from the 5' untranslated region of thyrotropin beta subunit mRNA and 60 nucleotides coding for an NH2-terminal precursor segment. This is followed by 339 nucleotides which code for the published amino acid sequence of the thyrotropin beta subunit. Following the 339 nucleotide beta subunit coding sequence, no termination codon is encountered for another 15 nucleotides. Thus, the cDNA codes for a thyrotropin beta subunit containing an additional 5 amino acids at the COOH terminus. The cDNA also contains 82 nucleotides of 3' untranslated sequence followed by a short poly(A) segment. Comparison of the bovine cDNA sequence to the recently described mouse thyrotropin beta subunit cDNA sequence reveals considerable homology throughout the coding sequence, including the COOH-terminal extension. These findings suggest the possibility that a thyrotropin beta subunit precursor is processed at both the NH2 and COOH termini.     

Gating properties conferred on BK channels by the beta3b auxiliary subunit in the absence of its NH(2)- and COOH termini

Both beta1 and beta2 auxiliary subunits of the BK-type K(+) channel family profoundly regulate the apparent Ca(2)+ sensitivity of BK-type Ca(2)+-activated K(+) channels. Each produces a pronounced leftward shift in the voltage of half-activation (V(0.5)) at a given Ca(2)+ concentration, particularly at Ca(2)+ above 1 microM. In contrast, the rapidly inactivating beta3b auxiliary produces a leftward shift in activation at Ca(2)+ below 1 microM. In the companion work (Lingle, C.J., X.-H. Zeng, J.-P. Ding, and X.-M. Xia. 2001. J. Gen. Physiol. 117:583-605, this issue), we have shown that some of the apparent beta3b-mediated shift in activation at low Ca(2)+ arises from rapid unblocking of inactivated channels, unlike the actions of the beta1 and beta2 subunits. Here, we compare effects of the beta3b subunit that arise from inactivation, per se, versus those that may arise from other functional effects of the subunit. In particular, we examine gating properties of the beta3b subunit and compare it to beta3b constructs lacking either the NH(2)- or COOH terminus or both. The results demonstrate that, although the NH(2) terminus appears to be the primary determinant of the beta3b-mediated shift in V(0.5) at low Ca(2)+, removal of the NH(2) terminus reveals two other interesting aspects of the action of the beta3b subunit. First, the conductance-voltage curves for activation of channels containing the beta3b subunit are best described by a double Boltzmann shape, which is proposed to arise from two independent voltage-dependent activation steps. Second, the presence of the beta3b subunit results in channels that exhibit an anomalous instantaneous outward current rectification that is correlated with a voltage dependence in the time-averaged single-channel current. The two effects appear to be unrelated, but indicative of the variety of ways that interactions between beta and alpha subunits can affect BK channel function. The COOH terminus of the beta3b subunit produces no discernible functional effects.